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Lung cancer organoid culture solution and culture reagent combination and culture method thereof

A culturing method and organoid technology are applied in the field of lung cancer organoid culture medium and the combination of culture reagents and culture, which can solve the problems of slow construction process, long time required, high time cost of lung cancer organoids, and achieve accelerated formation and growth. The effect of preventing contamination and shortening the enzymatic hydrolysis time

Active Publication Date: 2022-04-08
BEIJING DAXIANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the amount of cells obtained is also very small. In order to harvest more cells and conduct downstream experiments, it takes longer, which makes the construction process of the entire organ slow, resulting in high time costs for lung cancer organoid research. In addition, more The most important thing is that after a period of time in vitro culture of organoids, EMT transition will occur, and the mesenchymal properties in vivo will be gradually lost, which will be quite different from the real tumor properties in vivo

Method used

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  • Lung cancer organoid culture solution and culture reagent combination and culture method thereof
  • Lung cancer organoid culture solution and culture reagent combination and culture method thereof
  • Lung cancer organoid culture solution and culture reagent combination and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 protein conditioned medium

[0122] Protein-conditioned medium was prepared by the following methods:

[0123] S101. Resuscitate the target cell line, and use the first complete medium for culture. The first complete medium uses Advanced DMEM / F12+10%FBS (fetal bovine serum)+1% P / S (double antibody (penicillin plus streptomycin)). The target cell line is the overexpression cell line of the target protein, and the target gene is transferred into the CHO cell line by lentiviral transfection to realize the overexpression of the specific protein.

[0124] Target proteins include Wnt-3a, R-spondin1 and Noggin.

[0125] S102. After the initial culture until the cell confluence reaches 90%, pass to a 10cm culture dish, and then use the second complete medium for a screening culture for 2-3 days; after the cell confluence reaches 90%, pass to a new 10cm culture dish, and then use the second complete culture for secondary screening for 2 to 3 days. Wherein, the seco...

Embodiment 2

[0132] Example 2 Self-source conditioned medium

[0133] Self-sourced conditioned media were prepared using the following steps:

[0134] S201, performing physical pretreatment on the lung cancer sample to obtain sample tissue fragments;

[0135] S202. Perform enzymatic hydrolysis pretreatment on the sample tissue fragments, and filter to obtain tissue residues remaining after filtration;

[0136] S203. Spread the tissue residue obtained in step S202 on the bottom of the T25 bottle, and incubate the wall for 1-3 hours, then add medium (for example, DMEM basal medium + 5% serum + 3% PS), and cultivate;

[0137] S204. When cultured to the 4th day, observe whether there are fibroblasts crawling out of the tissue block, and perform the first liquid change; after that, change the liquid once every 3 days and observe, and collect the fibroblasts after a sufficient amount of fibrous cells grow out to obtain From tumor fibroblasts. The well-growing CAFs (tumor-associated fibroblast...

Embodiment 3

[0145] Example 3 Lung cancer organoid culture medium

[0146]A lung cancer organoid culture medium comprises a complete medium and a self-sourced conditioned medium, and the volume ratio of the complete medium to the self-sourced conditioned medium is 3:1. Wherein, the self-source conditioned medium adopts the self-source conditioned medium II obtained in Example 2.

[0147] Complete medium includes conditioned medium, basal medium, compound antibiotics and growth factors. Wherein, the volume ratio of conditioned medium and basal medium is 7:3, and in conditioned medium, by volume percentage, comprise Wnt-3a conditioned medium 20%, R-spondin1 conditioned medium 50% and Noggin conditioned medium 30% %, and the volume ratio of the three is 0.5:1.5:1 in turn. Basal media consisted of Advanced DMEM / F12, HEPES 10 mM and Glutamax 1x. That is, by volume ratio, Wnt-3a conditioned medium 20%, R-spondin1 conditioned medium 30%, Noggin conditioned medium 20%, basal medium 30%. Compou...

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Abstract

The invention relates to the technical field of biological tissue engineering, and discloses a lung cancer organoid culture solution and a culture reagent combination and a culture method thereof, and the lung cancer organoid culture solution comprises a complete medium and an autogenous condition medium; the complete culture medium comprises a conditioned culture medium, a basic culture medium, compound antibiotics and growth factors; the autogenous condition culture medium is obtained by culturing fiber cells extracted from lung cancer tissues by using a tumor-associated fibroblast culture medium. All the components of the culture solution have a synergistic effect, so that the formation and growth of lung cancer organoid cells are accelerated, and the culture speed is increased while the cell harvesting number is increased; and the in-vitro cultured organoid is more bionic, can be suitable for personalized culture of the lung cancer organoid, and can maintain the physical structure and pathological characteristics of the primary tissue of each patient.

Description

technical field [0001] The present application relates to the technical field of biological tissue engineering, for example, it relates to a lung cancer organoid culture medium, a combination of culture reagents and a culture method thereof. Background technique [0002] Lung cancer is one of the top ten malignant tumors in my country. At present, the etiology of lung cancer is not completely clear, and it is currently considered to be the result of the combined effects of environmental factors and genetic factors. Lung cancer organoids are of great importance to research. Although there are some research reports on the culture medium of lung cancer organoids, the growth of the culture process is slow, which increases the time input cost; and the bionicity is low. [0003] In the process of realizing the embodiment of the present application, it is found that there are at least the following problems in the related technology: in the culture process of the existing lung ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/071
Inventor 肖荣荣刘建闯李珮文孙艳廷张晓会周宇
Owner BEIJING DAXIANG BIOTECH CO LTD
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